Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Neutralization of mobile antiviral small RNA through peroxisomal import

Abstract

In animals, certain viral proteins are targeted to peroxisomes to dampen the antiviral immune response mediated by these organelles13. In plants, RNA interference (RNAi) mediated by small interfering (si)RNA is the main antiviral defence mechanism. To protect themselves against the cell- and non-cell autonomous effects of RNAi, viruses produce viral suppressors of RNA silencing (VSR)4, whose study is crucial to properly understand the biological cycle of plant viruses and potentially find new solutions to control these pathogens. By combining biochemical approaches, cell-specific inhibition of RNAi movement and peroxisome isolation, we show here that one such VSR, the peanut clump virus (PCV)-encoded P15, isolates siRNA from the symplasm by delivering them into the peroxisomal matrix. Infection with PCV lacking this ability reveals that piggybacking of these VSR-bound nucleic acids into peroxisomes potentiates viral systemic movement by preventing the spread of antiviral siRNA. Collectively, these results highlight organellar confinement of antiviral molecules as a novel pathogenic strategy that may have its direct counterpart in other plant and animal viruses.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: P15 efficiently sequesters 21 nt siRNAs.
Figure 2: Companion cell-specific P15 expression suppresses cell-to-cell SUL-silencing movement in a dose-dependent manner.
Figure 3: P15 and P15-bound siRNAs are imported into peroxisomes.
Figure 4: P15-mediated piggybacking of vsiRNAs potentiates PCV systemic spread.

Similar content being viewed by others

References

  1. Bender, S. et al. Activation of type I and III interferon response by mitochondrial and peroxisomal MAVS and inhibition by hepatitis C virus. PLoS Pathog. 11, e1005264 (2015).

    Article  Google Scholar 

  2. Ferreira, A. R. et al. Hepatitis C virus NS3-4A inhibits the peroxisomal MAVS-dependent antiviral signalling response. J. Cell. Mol. Med. 20, 750–757 (2016).

    Article  CAS  Google Scholar 

  3. Magalhaes, A. C. et al. Peroxisomes are platforms for cytomegalovirus’ evasion from the cellular immune response. Sci. Rep. 6, 26028 (2016).

    Article  CAS  Google Scholar 

  4. Incarbone, M. & Dunoyer, P. RNA silencing and its suppression novel insights from in planta analyses. Trends Plant Sci. 18, 382–392 (2013).

    Article  CAS  Google Scholar 

  5. Dixit, E. et al. Peroxisomes are signaling platforms for antiviral innate immunity. Cell 141, 668–681 (2010).

    Article  CAS  Google Scholar 

  6. Mohan, K. V., Som, I. & Atreya, C. D. Identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle. J. Virol. 76, 2543–2547 (2002).

    Article  CAS  Google Scholar 

  7. Deleris, A. et al. Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313, 68–71 (2006).

    Article  CAS  Google Scholar 

  8. Wang, X.-B. et al. The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative Argonautes in Arabidopsis thaliana. Plant Cell 23, 1625–1638 (2011).

    Article  CAS  Google Scholar 

  9. Carbonell, A. et al. Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24, 3613–3629 (2012).

    Article  CAS  Google Scholar 

  10. Chitwood, D. H. et al. Pattern formation via small RNA mobility. Genes Dev. 23, 549–554 (2009).

    Article  CAS  Google Scholar 

  11. Molnár, A. et al. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328, 872–875 (2010).

    Article  Google Scholar 

  12. Havelda, Z., Hornyik, C., Crescenzi, A. & Burgyan, J. In situ characterization of Cymbidium ringspot tombusvirus infection-induced posttranscriptional gene silencing in Nicotiana benthamiana. J. Virol. 77, 6082–6086 (2003).

    Article  CAS  Google Scholar 

  13. Dunoyer, P. et al. Identification, subcellular localization and some properties of a cysteine-rich suppressor of gene silencing encoded by peanut clump virus. Plant J. 29, 555–567 (2002).

    Article  CAS  Google Scholar 

  14. Dunoyer, P., Himber, C., Ruiz-Ferrer, V., Alioua, A. & Voinnet, O. Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways. Nat. Genet. 39, 848–856 (2007).

    Article  CAS  Google Scholar 

  15. Vargason, J. M., Szittya, G., Burgyan, J. & Hall, T. M. Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115, 799–811 (2003).

    Article  CAS  Google Scholar 

  16. Garcia-Ruiz, H. et al. Arabidopsis RNA-dependent RNA polymerases and Dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip Mosaic Virus infection. Plant Cell 22, 481–496 (2010).

    Article  CAS  Google Scholar 

  17. Azevedo, J. et al. Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein. Genes Dev. 24, 904–915 (2010).

    Article  CAS  Google Scholar 

  18. Leon, S., Goodman, J. M. & Subramani, S. Uniqueness of the mechanism of protein import into the peroxisome matrix: transport of folded, co-factor-bound and oligomeric proteins by shuttling receptors. Biochim. Biophys. Acta 1763, 1552–1564 (2006).

    Article  CAS  Google Scholar 

  19. Garcia-Ruiz, H. et al. Roles and programming of Arabidopsis ARGONAUTE proteins during Turnip Mosaic Virus infection. PLoS Pathog. 11, e1004755 (2015).

    Article  Google Scholar 

  20. Yelina, N. E., Savenkov, E. I., Solovyev, A. G., Morozov, S. Y. & Valkonen, J. P. Long-distance movement, virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families. J. Virol. 76, 12981–12991 (2002).

    Article  CAS  Google Scholar 

  21. Lazarow, P. B. Viruses exploiting peroxisomes. Curr. Opin. Microbiol. 14, 458–469 (2011).

    Article  CAS  Google Scholar 

  22. Bucher, E., Hemmes, H., de Haan, P., Goldbach, R. & Prins, M. The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants. J. Gen. Virol. 85(Pt 4), 983–991 (2004).

    Article  CAS  Google Scholar 

  23. Wan-Xiang, L. et al. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc. Natl Acad. Sci. USA 101, 1350–1355 (2004).

    Article  Google Scholar 

  24. Xie, Z., Allen, E., Wilken, A. & Carrington, J. C. DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 102, 12984–12989 (2005).

    Article  CAS  Google Scholar 

  25. Bechtold, N. & Pelletier, G. In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82, 259–266 (1998).

    CAS  PubMed  Google Scholar 

  26. Hamilton, A. J., Voinnet, O., Chappell, L. & Baulcombe, D. C. Two classes of short interfering RNA in RNA silencing. EMBO J. 21, 4671–4679 (2002).

    Article  CAS  Google Scholar 

  27. Liu, Y., Schiff, M. & Dinesh-Kumar, S. P. Virus-induced gene silencing in tomato. Plant J. 31, 777–786 (2002).

    Article  CAS  Google Scholar 

  28. Garcia, D., Garcia, S. & Voinnet, O. Nonsense-mediated decay serves as a general viral restriction mechanism in plants. Cell Host. Microbe. 16, 391–402 (2014).

    Article  CAS  Google Scholar 

  29. Hurkman, W. J. & Tanaka, C. K. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802–806 (1986).

    Article  CAS  Google Scholar 

  30. Lamattina, L., Gonzalez, D., Gualberto, J. M. & Grienenberger, J. M. Higher plant mitochondria encode an homolog of the nuclear-coded 30 kDa subunit of bovine mitochondrial complex I. Eur. J. Biochem. 217, 831–838 (1993).

    Article  CAS  Google Scholar 

  31. Qi, Y., Denli, A. M. & Hannon, G. J. Biochemical specialization within Arabidopsis RNA silencing pathways. Mol. Cell 19, 421–428 (2005).

    Article  CAS  Google Scholar 

  32. Garcia, D. et al. Ago hook and RNA helicase motifs underpin dual roles for SDE3 in antiviral defense and silencing of nonconserved intergenic regions. Mol. Cell 48, 109–120 (2012).

    Article  CAS  Google Scholar 

  33. Reumann, S. & Singhal, R. Isolation of leaf peroxisomes from Arabidopsis for organelle proteome analyses. Methods Mol. Biol. 1072, 541–552 (2014).

    Article  CAS  Google Scholar 

  34. Kräuter-Canham, R. et al. A transmitting tissue- and pollen-expressed protein from sunflower with sequence similarity to the human RTP protein. Plant Sci. 129, 191–202 (1997).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by a research grant from Agence Nationale de la Recherche (ANR-14-CE19-0014-01). It was also performed under the framework of the LABEX: ANR-10-LABX-0036_NETRNA and benefits from a funding from the state managed by the French National Research Agency as part of the Investments for the future program. We deeply thank S. Reumann for advice on plant peroxisome isolation and S. Bouzoubaa for advice on PCV purification.

Author information

Authors and Affiliations

Authors

Contributions

M.I. and P.D. designed the experiments. M.I. performed transgene construction, plant transformation and manipulation, viral purification and infection, immunoprecipitation and peroxisome isolation. M.I. and A.Z. performed RNA and protein extraction, RNA and protein gel blot analysis with the assistance of F.M. P.H. performed MS-MS protein analyses, while M.I. and M.E. performed immunohistochemistry. P.D. and M.I. wrote the manuscript and prepared the figures.

Corresponding author

Correspondence to P. Dunoyer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary References, Supplementary Figures 1–10, Supplementary Source Data, Supplementary Raw Data 1–10. (PDF 33861 kb)

Supplementary Table 1

Materials and Methods. (XLSX 49 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Incarbone, M., Zimmermann, A., Hammann, P. et al. Neutralization of mobile antiviral small RNA through peroxisomal import. Nature Plants 3, 17094 (2017). https://doi.org/10.1038/nplants.2017.94

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nplants.2017.94

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing